Suvorexant and mirtazapine improve chronic pain-related changes in parameters of sleep and voluntary physical performance in mice with sciatic nerve ligation

Both chronic pain and sleep disorders are associated with a reduction in the quality of life. They can be both a cause and a consequence of each other, and should therefore be simultaneously treated. However, optimal treatments for chronic pain-related sleep disorders are not well established. Here, we aimed to investigate the effects of suvorexant, a novel sleep drug, and mirtazapine, a noradrenergic and specific serotonergic antidepressant, on pain-related changes in sleep parameters in a preclinical chronic pain mice model, by partial sciatic nerve ligation. We evaluated the quantity, duration, and depth of sleep by analyzing the electroencephalogram and voluntary activity by counting the number of wheel rotations to determine various symptoms of sleep disorders, including reduced total sleep time, fragmentation, low quality, and impaired activity in the daytime. Suvorexant and mirtazapine normalized the reduction in sleep time and fragmented sleep, further regaining the sleep depth at sleep onset in the chronic pain state in nerve-ligated mice. Mirtazapine also increased the percentage of rapid eye movement sleep in mice. Suvorexant decreased voluntary activity, which was prolonged after administration; however, mirtazapine did not decrease it. Although the effects of suvorexant and mirtazapine on sleep and activity are different, both suvorexant and mirtazapine could be potential therapeutic agents for chronic pain-related sleep disorders.


Introduction
Chronic pain is pain that persists past normal healing time and is usually regarded as chronic when it lasts for more than 3 to 6 months [1]. Chronic pain often leads to further psychiatric disorders such as anxiety and depression, which significantly affect the health-related quality of life [2][3][4]. Sleep disorders are the most common psychiatric disorders related to pain [5][6][7][8] percentage of REM sleep, duration of each parameter, and time change in the power density of the δ wave during non-REM sleep were calculated from EEG/EMG. Experiment 3: Mice were habituated in a cage with a rotation wheel for 1 week and then underwent PSNL surgery. The number of wheel rotations was recorded 1 day before surgery and at 7, 14, and 21 days after surgery (Fig 1c). Experiment 4: Mice that underwent habituation and PSNL surgery similar to Experiment 3 were treated with suvorexant, mirtazapine, and their respective vehicles from day 7 to day 13 after surgery. The number of wheel rotations was recorded 1 day before surgery and at 6, 7, 13, 14, and 21 days after surgery (Fig 1d).

Neuropathic pain model
The mice were anesthetized with 3% isoflurane. We produced a PSNL model, as described previously [29]. A tight ligature ligated the right sciatic nerve with an 8-0 silk suture of approximately half of its diameter. In sham-operated mice, nerves were exposed without ligation.

Measurement of mechanical allodynia and thermal hyperalgesia
We performed the von Frey test to assess mechanical allodynia using automated von Frey equipment (Dynamic Plantar Aesthesiometer; Ugo Basile, Italy). The maximum force was set at 5 g to prevent tissue damage, and the ramp speed was 0.25 g/s to achieve an average baseline paw-withdrawal latency of 8-10 s in naive mice [32]. We performed the Hargreaves test to assess thermal hyperalgesia using a radiant heat light source (model 33 Analgesia Meter; IITC/ Life Science Instruments, USA). The intensity of the thermal stimulus was adjusted to achieve an average baseline paw-withdrawal latency of 8-10 s in naive mice [33]. The paw-withdrawal latency was determined as the average of four measurements per paw. Only quick hind paw movements (with or without licking of hind paws) away from the stimulus were considered a withdrawal response. Paw movements associated with locomotion or weight shifting were not considered responses. Before these tests, the mice were habituated for at least 20 min in a plastic cage on a metal grid bottom or in a clear acrylic cylinder (15 cm high and 8 cm in diameter) on a glass plate.

Sleep recordings
We recorded EEG/EMG to evaluate the sleep condition for 24 h on the last day of administration, as previously described [20,33]. Mice were mounted in a stereotaxic head holder and implanted with EEG and EMG electrodes for polysomnographic recordings (Pinnacle Technology, USA) under 3% isoflurane anesthesia. Two stainless steel EEG recording screws were positioned 1 mm anterior to the bregma or lambda, both 1.5 mm lateral to the midline. EMG activity was monitored using Teflon-coated steel wires placed bilaterally into both trapezius muscles. The collected data were analyzed using appropriate software (SLEEPSIGN Kissei Comtec, Japan). The vigilance of every 10 s epoch was automatically classified into three stages, that is, arousal, rapid eye movement (REM), and non-REM sleep; according to the standard criteria: 1) arousal was defined by a high EMG amplitude and low EEG amplitude; 2) REM sleep was defined by a low EMG amplitude, low EEG amplitude, and high θ wave activity; and 3) non-REM sleep was defined by low EMG amplitude, high EEG amplitude, and high δ wave activity [33]. Defined sleep-wake stages were visually examined, and corrected if necessary. Furthermore, we evaluated the time change in the power density of the δ wave to estimate the quality of sleep. The normalized power density of the δ wave was calculated as a value every 3 h for the total power density of the δ wave during non-REM sleep per day [33].

Assessment of voluntary activity
Wheel running was measured to assess the voluntary physical performance in a mouse model of neuropathic pain [34]. The mice were allowed to run freely on an open plastic wheel inside a standard cage (Melquest, Japan). Wheel rotations were electronically counted every 24 h and captured in a software program for data storage and analysis at various time points.

Statistics
All data are expressed as the mean ± standard error of the mean (SEM). Repeated-measures two-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparisons test was used to compare the paw withdrawal latency in von Frey and Hargreaves test (Fig 2), the percentage of each sleep time (Fig 3), the duration of wake, non-REM sleep, and REM sleep time (Fig 4), the normalized power density of δ wave among the "sham-vehicle," "PSNL-vehicle," and "PSNL-drug (suvorexant or mirtazapine)" groups on each time point (Fig 5), and the number of wheel rotations between "sham" and "PSNL" groups (Fig 6a). Repeated-measures one-way ANOVA followed by the Bonferroni multiple comparisons test was used to compare the number of wheel rotations at each time point in mice treated with each drug (Fig 6b and  6c). Statistical significance was set at P < 0.05. All statistical analyses were performed using Prism version 9.2. (GraphPad Software, La Jolla, CA, USA).

Effects of suvorexant and mirtazapine on pain behavior
The paw withdrawal latency in both the von Frey and Hargreaves tests decreased after nerve ligation (day 6) compared with baseline (day -1), as shown in Fig 2a- Fig 2a); however, the effect did not last until the next day. There was no significant difference in the response to thermal stimuli (Fig 2b). . These data suggest that mirtazapine improved PSNL-induced pain behaviors, but the improvement with suvorexant was partial.

Effects of suvorexant and mirtazapine on the amount of sleep time
We analyzed EEG/EMG in PSNL mice to determine the effects of drugs on the amount of sleep time. We calculated the time change every 2 h of the percentage of sleep (Fig 3a and 3d) and compared the values added up in the light and dark phases (Fig 3b and 3e). The PSNLvehicle group showed a significant decrease in the percentage of sleep during the light phase, which is the rest period for mice. Suvorexant and mirtazapine significantly normalized the reduction in the percentage of sleep to the same level as the sham group (PSNL-vehicle vs. PSNL-suvorexant: 51.8% vs. 76.3%, P = 0.0007; PSNL-vehicle vs. PSNL-mirtazapine: 47.0% vs. 74.1%, P = 0.0050) during the light phase in PSNL (Fig 3b and 3e). In addition, mirtazapine increased the proportion of REM sleep per total sleep (Fig 3f), but not suvorexant (Fig 3c). The proportion of REM sleep was calculated based on the amount of REM sleep and non-REM sleep time (S1 Fig).

Effects of suvorexant and mirtazapine on the duration time of wakefulness and sleep
Mice are polyphasic sleepers, alternating between sleep and wakefulness in the order of minutes [35]. We calculated the mean duration of wakefulness, non-REM sleep, and REM sleep every 6 hours based on recorded EEG to assess the duration of wakefulness and sleep. The sham mice showed long durations of arousal during the dark phase, which is an active period for mice. However, PSNL mice showed a significant reduction in the duration of wakefulness

Effects of suvorexant and mirtazapine on the normalized power density of δ waves
To assess the change in the depth of sleep, we analyzed the power density of δ waves, which are the factors that define the depth of sleep. The power density of the δ wave during non-REM sleep was calculated every 3 h, and the ratio with the average value during the entire day was calculated for each individual. In the sham group, the power density of the δ wave in non-REM sleep was particularly strong during the early light phase. The δ power tended to weaken from the late light phase to the early dark phase and became stronger again toward the latter

Effects of suvorexant and mirtazapine on the voluntary activity
To assess voluntary activity, we placed the mice in cages with a running wheel and recorded the number of wheel rotations. PSNL significantly reduced the wheel rotation compared to sham (sham vs. PSNL: 18,650/day vs. 11,801/day, P = 0.0040 at day 7; 19,010/day vs. 11,500/ day, P = 0.0015 at day 17; Fig 6a). Suvorexant and mirtazapine were administered after PSNL surgery, and the wheel rotation was evaluated at certain test points, as shown in Fig 6b. Suvorexant treatment reduced the number of wheel rotations compared with the baseline. On the other hand, mirtazapine treatment did not change the number of wheel rotations (Fig 6c). These results suggest that mirtazapine improves voluntary activity in PSNL mice.

Discussion
Recent clinical and basic research suggest that sleep disorder is a heterogenic disorder and requires multifaceted evaluation to correctly determine the state of sleep and the effects of therapeutic agents [36][37][38]. We evaluated the sleep status of a preclinical pain model to be close to clinical situations by examining the amount of sleep time, sleep duration, sleep depth, and the effect on daytime activity objectively using EEG or wheel rotation. PSNL caused a decrease in REM and non-REM sleep time based on EEG analysis (Fig 3). In addition, we found that the duration of non-REM sleep was interrupted, resulting in fragmented sleep in the neuropathic pain model (Fig 4). The duration of wakefulness during the dark phase was substantially reduced in the neuropathic pain model mice compared with that in the sham group (Fig 4). Non-REM sleep is classified into stages based on depth, which correlates with the strength of the δ power density. The sham group showed deep sleep in the early stages of the light phase with a high power density of δ wave, following which the δ power gradually became shallower towards the late stages of the light phase. In contrast, such early deep sleep was not observed in the PSNL vehicle group (Fig 5). Furthermore, PSNL reduced the number of wheel rotations (Fig 6), indicating that sleep was shortened and fragmented, the rhythm of sleep depth was lost, and arousal and voluntary activity were impaired due to chronic pain caused by nerve injury.
The orexin receptors (orexin-1 and orexin-2 receptor) are involved in stabilizing arousal and suppressing sleep by maintaining the activity of monoaminergic neurons in the central arousal system [21,22]. Suvorexant could be a potential therapeutic agent to improve painrelated sleep disorders because it causes pharmacological inhibition of orexin receptors [23,24]. In the present study, suvorexant improved chronic pain-related changes in sleep parameters. Suvorexant improved sleep time by increasing non-REM and REM sleep in the chronic neuropathic pain model (Fig 3). Suvorexant also improved the fragmentation of non-REM sleep, non-sustainability of arousal (Fig 4), and loss of rhythm in sleep depth (Fig 5). However, suvorexant did not improve the voluntary activity (Fig 6). Orexin can potentiate the excitatory synaptic transmission of dopaminergic neurons in the mesolimbic system, causing motivation [39]. Furthermore, the mesopontine tegmentum, including structures relevant to locomotion and muscle tone, is also a major target of orexin [39]. These findings suggest that blocking orexin signaling may lead to lower locomotive behavior.
Several studies have shown that mirtazapine, which is commonly used clinically, may improve sleep disorders [26,27,40,41]. The sedative effects of mirtazapine are thought to be mediated by suppressing histaminergic and serotonergic neurons in the central arousal system because mirtazapine shows strong antagonistic effects on the H 1 histamine receptor and 5HT 2 serotonin receptor [27]. The effects of mirtazapine on the endocrine system by normalizing the hypothalamo-pituitary-adrenocortical system overactivation and blunting the melatonin system could be a potential mechanism for sleep improvement [41]. A previous study showed that 1 mg/kg of mirtazapine improves only sleep quantity in PSNL mice, but no other symptoms have been observed [42]. Analysis of multiple parameters of sleep revealed that mirtazapine improved the reduction in sleep time, sleep fragmentation, and depth of sleep onset. The results of the wheel rotation experiment showed an improving trend in the decline of involuntary movement when PSNL mice were treated with mirtazapine. Mirtazapine was reported to promote active motion and have anti-immobility effects in a rat forced swimming test [43]. The enhancing effects of mirtazapine on motivation may be related to increased dopamine release in the frontal cortex [44,45]. However, in a clinical trial, up to 54% of patients reported daytime sleepiness as an adverse effect of mirtazapine [40]. Mirtazapine predominantly produces anti-histaminergic effects at lower doses, whereas noradrenergic effects become more predominant at higher doses [46]. Due to this unique pharmacological profile, mirtazapine is thought to produce relatively more sedation at lower doses.
In this study, mirtazapine significantly increased the REM sleep time in PSNL. The effect of the amount of REM sleep on animal health remains unclear, and further research is needed. A recent study on REM sleep and mortality showed that for every 5% decrease in REM sleep, mortality increased by 13% [47]. Strategies to preserve REM sleep may reduce the mortality risk in patients with reduced REM sleep. On the other hand, some antidepressants that increase REM sleep are associated with nightmares and REM sleep behavior disorder as side effects [48] because nightmares are essentially a REM pathology [49]. However, there is no consistent view on the effect of mirtazapine on REM sleep, with both a report that mirtazapine increases REM sleep [50] and that it does not change [51].
We investigated the effects of suvorexant and mirtazapine on chronic pain-related changes in sleep parameters and voluntary physical performance in a preclinical model. However, the specific neurological mechanisms by which these agents regulate chronic pain-related sleep disorders require further investigation.